Antitumorigenic lactone derivative

ABSTRACT

There are disclosed novel compounds which are extracted with an organic solvent from Eupatorium sachalinense belonging to the genus Eupatorium and isolated by normal separating and refining procedures, the compounds being expressed by the general formula: ##STR1## where R is ##STR2## a pale yellowish oily substance having an optical rotation of [α] D   24  -121° (C=O.75, ethanol), a molecular weight of 420 estimated from peak value in mass spectrum measurement (theoretical value: 420), a molecular formula of C 22  H 28  O 8 , an end absorption at 210 mm (ε:14800) in ultraviolet absorption spectrum, peaks in infrared absorption spectrum by liquid film method at the frequencies (cm -1 ) of 3400, 2920, 2845, 1760, 1740, 1705, 1658, 1440, 1370, 1325, 1220-1280, 1180, 1133, 1107, 1070, 1020, 970, 881, 840, 819, 788, 758, 710, 662, 630, 607 and 582, assignments of respective protons in NMR spectrum (δ,CDCl 3 ) at C 6  --H=5.96 (1H, dd, J=11,2), C 4  --CH 3  =1.80 (3H, d, J=1), C 10  --CH 3  =1.84 (3H, s), C 3&#39;  --H=6.90 (1H, t, J=5.5), C 4&#39;  --H=4.37 (2H, d, J=5.5), C 5&#39;  --H=4.31 (2H, s), exo CH 2  =5.79,6.33 (1H, d, J=2), COCH 3  = 2.12 (3H, s); ##STR3## or [III] hydrogen; and a process for the preparation of these compounds. These compounds are found to have not only inhibitive effects, even in a low concentration, against experimental cancer cells (HeLa) but also considerable life prolonging effects in in vitro experiments on mice inoculated with Ehrlich ascites, suggesting similar effects on malignant tumors in humans.

BACKGROUND OF THE INVENTION

This invention relates to novel compounds expressed by the following general formuula: ##STR4## where R is ##STR5## a pale yellowish oily substance having an optical rotation of [α]_(D) ²⁴ -121° (C=0.75, ethanol), a molecular weight of 420 estimated from peak value in mass spectrum measurement (theoretical value: 420), a molecular formula of C₂₂ H₂₈ O₈, an end absorption at 210 mm (ε:14800) in ultraviolet absorption spectrum, peaks in infrared absorption spectrum by liquid film method at the frequencies (cm⁻¹) of 3400, 2920, 2845, 1760, 1740, 1705, 1658, 1440, 1370, 1325, 1220-1280, 1180, 1133, 1107, 1070, 1020, 970, 881, 840, 819, 788, 758, 710, 662, 630, 607 and 582, assignments of respective protons in NMR spectrum (δ, CDCl₃) at C₆ --H=5.96 (1H, dd, J=11,2), C₄ --CH₃ =1.80 (3H, d, J=1), C₁₀ --CH₃ =1.84 (3H, s), C_(3') --H=6.90 (1H, t, J=5.5), C_(4') -- H=4.37 (2H, d, J=5.5), C_(5') --H=4.31 (2H, s), exo CH₂ =5.79,6.33 (1H, d, J=2), COCH₃ =2.12 (3H, s); ##STR6## or [III] hydrogen; and a process for the preparation of these substances.

Recently, analytical research into the components of the various plants belonging to the genus Eupatorium has been pursued by Kupchan et al (J. Org. Chem. 38, 1260, 2189 (1973)), and various compounds having a significant antitumorigenic effect have been isolated. Among these compounds, those having the germacrane skeleton include the following: ##STR7## and it is known that these compounds show an inhibitory effect against growth of the cancer cells in the in vitro tests on the experimental cancer cells.

As a result of many experimental separation and refining of the extracts from Eupatorium sachalinense, a plant belonging to the genus Eupatorium, the present inventors have succeeded in isolating three novel substances (I, II and III) of the germacrane system, and it was clarified from various spectral analytical data and chemical reactions that these substances have the following chemical structures:

    ______________________________________                                          ##STR8##                                                                       ##STR9##              (Hiyodorilacton-A)                                       ##STR10##             (Hiyodorilacton-B)                                      III : R = H            (Hiyodorilacton-C)                                      ______________________________________                                    

The compound I obtained from the plants belonging to Eupatorium is a stereoisomer of eucannabinolide which is described by B. Drozdz et al (Collection Czechoslov Chem. Commun. 37 1546 1972)) and by M. Holub et al (ibid 42 1053 (1977)) and of eupaformosanin which was described by Kuo-Hsing et al (Phytochemistry 16 1068 (1977)), eucannabinolide and eupaformosanin having the structures as indicated below. ##STR11##

Compound I is same as the compounds IV and V in plane structure but has the acetoxyl group on the mother ring in beta-position in contrast the alpha location of the counterpart in compound V.

Upon comparing the physical properties of compound I with the corresponding data for compound IV given by B. Drozdz et al, the two compounds are found to have same appearance, optical rotation and molecular weight and exhibit peaks at similar frequencies in their infrared absorption spectra. However, in NMR spectrum, the hydrogen signal of the methylene group at C--4' of the side chain appears at δ4.37 (d, J=5.5) with compound I, in contrast to δ4.00 (d, J=6.0) of compound IV.

Compounds I and IV are identical with respect to the structure of the mother ring but they are considered to be stereoisomers with respect to the side chain.

Although it is not known whether the compounds IV and V have anticarcinogenic activity, the compounds I to III according to the present invention have been ascertained to have not only high inhibitory effect against growth of experimental cancer cells (HeLa) when used even in a low concentration, but also considerable life prolonging effect in the in vitro experiments on mice inoculated with Ehrlich ascites cancer cells, suggesting availability of these substances as a medicine for malignant tumors in humans.

The compounds I to III are contained throughout the body of the plant including root, stems and leaves, but since they exist in greater concentration in the leaves, it is advantageous to use the leaves as their extraction source.

SUMMARY OF THE INVENTION:

It is an object of the present invention to provide the novel compounds mentioned above and a process for the preparation thereof.

According to the present invention, there is provided a compound expressed by the general formula: ##STR12## where R is ##STR13## a pale yellowish oily substance having an optical rotation of [α]_(D) ²⁴ -121° (C=0.75, ethanol), a molecular weight of 420 estimated from peak value in mass spectrum measurement (theoretical value:420), a molecular formula Of C₂₂ H₂₈ O₈, an end absorption at 210 mm (ε:14800) in ultraviolet absorption spectrum, peaks in infrared absorption spectrum by liquid film method at the frequencies (cm⁻¹) of 3400, 2920, 2845, 1760, 1740, 1705, 1658, 1440, 1370, 1325, 1220-1280, 1180, 1133, 1107, 1070, 1020, 970, 881, 840, 819, 788, 758, 710, 662, 630, 607 and 582, assignments of respective protons in NMR spectrum (δ, CDCl₃) at C₆ --H=5.96 (1H, dd, J=11,2), C₄ --CH₃ =1.80 (3H, d, J=1), C₁₀ --CH₃ =1.84 (3H, s), C_(3') --H=6.90 (1H, t, J=5.5), C_(4') -- H=4.37 (2H, d, J=5.5), C_(5') --H=4.31 (2H, s), exo CH₂ =5.79,6.33 (1H, d, J=2), COCH₃ =2.12 (3H, s); ##STR14## or [III] hydrogen.

According to the invention, there is also provided a process for the preparation of the compound mentioned above, comprising extracting the compound with organic solvent from Eupatorium sachalinense belonging to the genus Eupatorium, followed by separation and refining.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 3 are infrared spectra of compounds I to 3, respectively.

The present invention will be illustrated more particularly by the following experiments and Example.

EXPERIMENTAL EXAMPLE 1

(a) Method of experiment

The roots (0.3 kg by half-dry weight), stalks (1.6 kg by half-dry weight) and leaves (2.1 kg by half-dry weight) were ground and extracted respectively with methanol. Each liquid extract was filtered and the filtrate was concentrated under reduced pressure to obtain an oily residue containing the object compounds.

(b) Results

The amounts (weight) of the oily materials obtained from the respective parts of the plant (Eupatorium sachalinense) and their effect against growth of the HeLa Cells were as shown in Table 1 below.

                  Table 1                                                          ______________________________________                                         Extraction source                                                                         Weight (g)                                                                               Concentration                                                                              Inhibitory effect                             ______________________________________                                         Root       12        200 μg/ml                                                                                12.5%                                        Stalk      47        200 μg/ml                                                                               12.5%                                         Leaf       99         35 μg/ml                                                                               50%                                           ______________________________________                                    

For producing the object substances from Eupatorium sachalinense according to the process of this invention, first the dried portions of Eupatorium sachalinense are extracted with an organic solvent at normal temperature or with heating and the liquid extract is concentrated under reduced pressure to obtain the primary extract.

The organic solvent used here may be, for example, an alcohol such as methanol, ethanol or propanol, a ketone such as acetone, methyl ethyl ketone or methyl isobutyl ketone, a halogenated hydrocarbon such as ethylene dichloride, chloroform or carbon tetrachloride, an aromatic hydrocarbon such as benzene, toluene or xylene, or an ester such as methyl acetate or ethyl acetate. However, since the dried portions of the plant used for the extraction still contain water, it is advantageous to use a volatile solvent miscible with water. Considering these conditions and economical availability, it is most advantageous to employ a hydrophilic organic solvent such as a lower alcohol or acetone, most preferably methanol.

Then the primary extract is cleared of the hydrophilic or non-hydrophilic impurities by means of solvent fractionation. As the solvent system used for this treatment, it is recommended to employ a suitable combination of a hydrocarbon or halogenated hydrocarbon with water, aqueous methanol or aqueous acetone.

For instance, as described in the Examples shown later, the primary extract is first mixed with chloroform and water and the fractionated chloroform layer is concentrated under reduced pressure to eliminate the hydrophilic impurities, and then in order to remove the non-hydrophilic impurities, the concentrate is distributed in a petroleum ether and a 10% aqueous methanol solution and the fractionated aqueous methanol layer are similarly concentrated, thereby obtaining a concentrate containing the object compounds. The latter operation may be accomplished by washing the primary extract with petroleum ether, but in this case, the refining efficiency is poor.

For separating the respective components from the thus obtained secondary extract, it is generally recommended to employ column chromatography using silica gel or alumina as adsorbent, but in some cases the object may be attained by using a simple batch type adsorption-elution method. It is also possible in certain cases to apply the adsorption method by omitting a part of the primary or secondary extraction process.

The eluent used for column chromatography is preferably a non-hydrophilic solvent such as chloroform, ethyl acetate ester, dichloroethane or benzene, which, if need be, is mixed with a small quantity of hydrophilic solvent such as methanol or acetone. In this case, it is advisable for obtaining better separation efficiency to gradually increase the ratio of the hydrophilic solvent to the non-hydrophilic solvent during the eluting treatment.

For attaining perfect separation of each component, it is suggested that chromatography be repeated for a particular fraction.

Finally, each fraction providing a single spot on the thin-layer chromatograph is fractionated and further concentrated and dried under reduced pressure, thereby obtaining the respective object components, compounds I, II and III.

The chemical properties and other characterizations of the object compounds of this invention are shown in Tables 2 and 3 below.

                                      Table 2                                      __________________________________________________________________________     External appearance, optical rotation, etc.                                                        Molecular weight                                                                        Ultraviotet                                       Compound                                                                             External                                                                             Optical rotation                                                                       measured by mass                                                                        absorption                                        No.   appearance                                                                           [α].sub.D.sup.24 Ethanol                                                         spectrum spectrum                                                                             Infrared absorption spectrum                __________________________________________________________________________           Colorless                                                                            -121°                                                                           Estimate End asorp-                                                                           3400                                                                              2920                                                                              2845                                                                              1760                                                                              1740 1705                                                                              1658                          ˜ Pale                                                                         (C = 0.75)                                                                             from frag-                                                                              tion                                                    yellow        ment peak                                                                            : 420    1440                                                                              1370                                                                              1325                                                                              1220                                                                              ˜1280                                                                         1180                                                                              1133                                                 210 nm                                            I     Oily          Theore-  1107  1070                                                                              1020                                                                               970                                                                               881                                                                               840  819                                           tical : 420                                                                             (ε:14800)                                                                     788                                                                               758                                                                               710                                                                               662                                                                               630  607                                                                               582                                        C.sub.22 H.sub.28 O.sub.8  (FIG. 1)                              Colorless                                                                            -140°                                                                           Found : 404                                                                             End absorp-                                                                          3460                                                                              2940                                                                              2850                                                                              1760                                                                              1750 1720                                                                              1660                          ˜Pale                                                                          (C = 0.67)       tion                                                    yellow        Theore-        1440                                                                              1375                                                                              1324                                                                              1225                                                                              ˜1235                                                                         1180                                                                              1138                    II                  tical : 404                                                                             210 nm                                                  Oily                         1110                                                                              1075                                                                              1020                                                                               970                                                                               883  840                                                                               820                                        C.sub.22 H.sub.28 O.sub.7                                                               (ε:30400)                                                                     790                                                                               755                                                                               710                                                                               667                                                                               630  608                                                                               585                                                                   (FIG. 2)                              Colorless                                                                            -109°                                                                           Found : 306                                                                             End absorp-                                                                          3460                                                                              2920                                                                              2860                                                                              1760                                                                              1740 1660                                                                              1450                          ˜Pale                                                                          (C = 0.91)       tion                                                    yellow        Theore-        1435                                                                              1370                                                                              1328                                                                              1280                                                                              1250 1180                                                                              1140                     III                tical : 304                                                                             210 nm                                                  Oily                         1110                                                                              1060                                                                              1033                                                                               962                                                                               940  880                                                                               840                                        C.sub.17 H.sub.22 O.sub.5                                                               (ε:14600)                                                                     817                                                                               790                                                                               753                                                                               710                                                                               664  625                                                                               607                                                        580  .     (FIG. 3)                        __________________________________________________________________________

                                      Table 3                                      __________________________________________________________________________     NMR spectrum: assignment of respective proton (δ)                         ##STR15##                                                                     Compound                                   Exo                                 No.   C.sub.6H                                                                             C.sub.4CH.sub.3                                                                     C.sub.10CH.sub.3                                                                     C.sub.3'H                                                                           C.sub.8H                                                                            C.sub.4'H                                                                           C.sub.5'H                                                                           CH.sub.2                                                                            COCH.sub.3                     __________________________________________________________________________            5.96 1.80 1.84  6.90      4.37 4.31 5.79 2.12                           I     (1H, dd,                                                                             (3H, d,                                                                             (3H, s)                                                                              (1H, t,   (2H, d,                                                                             (2H, s)                                                                             6.35 (3H, s)                              J = 11, 2)                                                                           J = 1)     J = 5.5)  J = 5.5)  (1H, d,                                                                        J = 2)                                    5.98  1.80       6.81      4.31 1.80 5.79 2.10                           II    (1H, dd,                                                                             (6H,s)     (1H, t,   (2H, d,                                                                             (3H, s)                                                                             6.36 (3H, s)                              J = 11, 3)       J = 6)    J = 6)    (1H, d,                                                                        J = 2)                                    5.86  1.80 1.90       4.20           5.70 2.07                           III   (1H, dd                                                                              (3H, d,                                                                             (3H, s)    (1H, m,        6.38 (3H, s)                              J = 11, 2)                                                                           J = 1.5)        w 1/2 = 9)     (1H, d,                                                                        J = 2)                              __________________________________________________________________________

(1) Inhibitory effect against HeLa S₃ cells

(a) Method of experiment

The HeLa S₃ cells were spread on 6 cm-diameter plastic laboratory plates in the amount of 0.7×10⁵ cells/plate and cultured by using an MEM medium containing 60 μg/ml of kanamycin and 10% calf serum. 2 days later, the medium was replaced with one containing the compounds of this invention. The cell population at that time was 1.58×10⁵ cells/plat. After addition of the compounds, the cells were cultured for three days, and then, after removing the medium, the culture was washed with a physiological saline solution, treated with 0.05% trypsin at room temperature for about 15 minutes, and then pipetted and dispersed. The number of surviving cells was counted by a coal-tar counter, and percent inhibition was measured by counting the number of the living cells in the control section to determine ID₅₀. ##EQU1##

(b) Results

The results of the experiment were as shown in Table 4.

                  Table 4                                                          ______________________________________                                         Inhibitory effect against                                                      growth of HeLa cells                                                           Compound  Centration  Percent in- ID.sub.50                                    No.       (μg/ml)  bition (%)  (μg/ml)                                   ______________________________________                                                   4           100                                                      I         2           74.9        1.4                                                    1           29.4                                                               1.5         100                                                      II        0.75        57.0        0.62                                                   0.375       28.4                                                     ______________________________________                                    

(2) Life-prolonging effect for mice suffering from Ehrlich ascites cancer

(a) Method of experiment

The mice of ICR/JCL species (♂, 5-week-old, 5 mice per group) were intraperitoneally inoculated with 2,000,000 pieces of Ehrlich ascites cancer cells, and the compounds of this invention were administered, starting 2 hours after inoculation, in the dosages shown in Table 5 below once a day for a total period of 10 days. The mice were observed for 50-days.

(b) Results

The experimental results were as shown in Table 5 below.

                  Table 5                                                          ______________________________________                                         Life-prolonging effect for the                                                 mice suffering from Ebrlich                                                    ascites cancer                                                                 Compound    Dose          Life prolongation                                    No.         mg/kg         rate*                                                ______________________________________                                         I           7.50          253                                                              15.0          232                                                  II          7.50          159                                                              15.0          173                                                  III         7.50          95                                                               15.0          108                                                  ______________________________________                                          *The "life prolongation rate" shows the average number of survival days o      the drug treated groups as calculated with the average number of survival      days of the control group being indexed as 100.                          

As apparent from the results shown in Table 4 and Table 5, the compounds I and II of this invention manifested not only a high inhibitory effect against growth of the cancer cells, with a low concentration (dose), in the in vitro tests on the experimental cancer (HeLa) cells but also a prominent life-prolonging effect for the mice afflicted with Ehrlich ascites cancer but administered with the compounds of this invention in comparison with the control mice. These results are suggestive of utility of these compounds for treatment of tumors in humans.

Although compound III is not so conspicuous in effect as the compounds I and II, it is useful as an intermediate intended to be transformed into the compounds I and II and associated compounds.

Now the process of this invention is described in further detail by way of the following examples.

EXAMPLE 1

The leaves of Eupatorium sachalinense (2.1 kg by half-dry weight) collected in August at Kita Saku-gen, Nagano-ken, were ground and then extracted with 1.2 liters of methanol divided in three portions. The exudates from the respective maceration treatments were filtered and the filtrates were combined and concentrated under reduced pressure to obtain 99 gr of an oily residue.

99 gr of this extract was divided into two layers with 3 liters of water and 4.4 liters of chloroform, and the solvent was distilled off from the chloroform layer under reduced pressure to obtain 30 gr of residue. This residue was dissolved in a mixed solution of 3.9 liters of petroleum ether and 2.9 liters of methanol/water (9/l) mixture, and after agitation, the methanol layer was fractionated. This methanol solution was concentrated under reduced pressure and water was removed from the residual solution by freeze-drying to obtain 7.8 gr of residue.

6.6 gr of this residue was dissolved in chloroform and the solution was passed through a column packed with 1 kg of silica gel to adsorb the object compound, and this was followed by elution first with chloroform, then with a chloroform-methanol solution and finally with methanol. The elution with a chloroform-methanol solution was performed by gradually increasing the methanol ratio.

(a) Of the eluted fraction, only the portion which had an Rf value of thin-layer chromatography (TLC) of 0.4 (using a chloroform (10)--methanol (1) developing solution) was collected and that portion was concentrated under reduced pressure and evaporated to dryness.

1.9 gr of the obtained residue was dissolved in a chloroform/acetone (4/l) mixed solution and passed through a column packed with 1.2 kg of silica gel to adsorb the object compound, and the adsorbate was eluted using a chloroform/acetone eluting solution whose chloroform:acetone ratio was gradually increased from an initial 4:1 to final 2:1, and the fraction containing the eluted-out object compound was gathered, concentrated under reduced pressure and dried.

There was consequently obtained 1.4 gr of a colorless oily substance (compound I) having the following properties: [α]_(D) ²⁴ (EtOH, C=0.75):-121°; IR (liquid film) cm⁻¹ :3400, 1760, 1740, 1705, 1658.

(b) The fraction portion having a TLC Rf value of 0.6 (using a chloroform (10)/methanol (1) developing solution) was collected, concentrated under reduced pressure and evaporated to dryness, and 1.4 gr of this residue was dissolved in a small amount of a chloroform/acetone (10/l) mixed solution and passed through a column packed with 1 kg of silica gel to adsorb the object compound, and the adsorbate was eluted using a chloroform/acetone (10/l) eluting solution, whereby a fraction containing compound II (TLC Rf: 0.6 (using a chloroform (5)/acetone (2) developing solution)) and then a fraction containing compound III (TLC Rf: 0.7 (using a chloroform (5)/acetone (2) developing solution)) were eluted out. These fractions were concentrated under reduced pressure to obtain 0.9 gr of a colorless oily product of compound II ([α]_(D) ²⁴ (EtOH, C=0.67):-140°; IR (liquid film) cm⁻¹ :3460, 1760, 1750, 1720, 1660) and 0.17 gr of a colorless oily product of compound III ([α]_(D) ²⁴ (EtOH, C=0.91):-109°; IR (liquid film) cm⁻¹ :3460, 1760, 1740, 1660). 

What is claimed is:
 1. Hiyodorilacton-B having the formula: ##STR16## 